title: Three-dimensional reconstruction of nucleolar components by electron microscope tomography.
abstract: The nucleus is a complex volume constituted of numerous subcompartments in which specific functions take place due to a specific spatial organization of their molecular components. To understand how these molecules are spatially organized within these machineries, it is necessary to investigate their three-dimensional organization at high resolution. To reach this goal, electron tomography appears to be a method of choice; it can generate tomograms with a resolution of a few nanometers by using multiple projections of a tilted section several hundred to several thousand nanometers in thickness imaged by transmission electron microscopy (TEM).Specific identification of molecules of interest contained within such thick sections requires their specific immunocytochemical labelling using electron-dense markers. We recently demonstrated that electron tomography of proteins immunostained with nanogold particles before embedding, and subsequently amplified with silver, was very fruitful due to the inherently high spatial resolution of the medium-voltage scanning and transmission electron microscope (STEM). Here we describe this approach, which is very efficient for tracing the 3D organization of proteins within complex machineries by using antibodies raised against one of the proteins, or against GFP to analyse GFP-tagged proteins.
authors.affiliation: Department of Morphology, Institute of Biology, Tbilisi State University, Tbilisi, Georgia.
authors.forename: Pavel
authors.initials: P
authors.lastname: Tchelidze
authors.forename: Hervé
authors.initials: H
authors.lastname: Kaplan
authors.forename: Adrien
authors.initials: A
authors.lastname: Beorchia
authors.forename: Marie-Françoise
authors.initials: MF
authors.lastname: O'Donohue
authors.forename: Hélène
authors.initials: H
authors.lastname: Bobichon
authors.forename: Nathalie
authors.initials: N
authors.lastname: Lalun
authors.forename: Laurence
authors.initials: L
authors.lastname: Wortham
authors.forename: Dominique
authors.initials: D
authors.lastname: Ploton
mesh_terms: D045744:Cell Line, Tumor; D002466:Cell Nucleolus / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D055032:Electron Microscope Tomography / Q000379:methods*; D006046:Gold / Q000737:chemistry*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods; D021621:Imaging, Three-Dimensional; D008175:Lung Neoplasms / Q000473:pathology; D053768:Metal Nanoparticles / Q000737:chemistry*; D018613:Microscopy, Confocal / Q000379:methods; D046529:Microscopy, Electron, Transmission / Q000379:methods; D036103:Nanotechnology / Q000379:methods*; D013696:Temperature
chemical_list: D049452:Green Fluorescent Proteins; D006046:Gold